H5N1 is a highly pathogenic avian influenza virus that can cause severe disease and death in humans. H5N1 is spreading rapidly in bird populations world-wide and there is great concern that this virus will begin to transmit between people and cause a global pandemic catastrophe. Vaccines are the cornerstone strategy for combating avian flu but there are numerous challenges in producing the vast numbers of safe and effective doses that are needed to prevent infection and to save millions of lives. One previous effort to eliminate traditional egg-based vaccine production involved a clinical trial that tested baculovirus-derived recombinant H5 Hemagglutinin (rH5). The results showed that rH5 was safe but could only stimulate "protective" antibodies in ~50% of subjects after 2 injections. We believe the relative ease in manufacturing rH5 protein could be a very important strategy for addressing the world's vaccine stockpiling efforts if rH5 immunogenicity was improved. We also believe this is possible by combining rH5 with an innovative adjuvant that targets dendritic cell function. Here we describe straightforward proof of concept experiments where rH5 will be formulated with an oil-in-water emulsion and Glucopyranosyl Lipid A (GLA), a synthetic Toll-Like Receptor-4 (TLR-4) agonist. Collectively, these components augment antibody responses needed for preventing viral infection, as well as stimulate T cells that limit disease progression within the host and reduce viral transmission within the population. The improved immunogenicity of this pandemic vaccine will be established following completion of three specific aims. We will produce, characterize and optimize vaccine formulations for physical integrity, stability, and GLA potency using mouse and human DC activation assays. Improvements in vaccine immunogenicity will be established in mice by measuring antigen-specific antibody responses, including those that can cross-react and neutralize H5N1 virus from different strains. Identification of a lead formulation for subsequent work will require that the GLA adjuvant can;(1) mediate a significant dose-sparing effect and maintain maximal antibody responses following a 10-fold reduction in rH5 concentration, and (2), induce long- lasting CD4+ and CD8+ T cell responses in mice. If successful, then rH5+GLA adjuvant will be tested in virus challenge models by inoculating mice and ferrets with two strains of H5N1. Viral load, disease progression and survival will be monitored, as well as adjuvant dose-sparing activity and induction of cross-reactive antibodies. Our criteria for vaccine efficacy is that GLA adjuvant will stimulate 100% survival at 1/10 the rH5 dose compared to nonadjuvanted rH5 vaccine. Moreover, the adjuvant will be cross-protective and stimulate significant survival in animals challenged with a different H5N1 virus. Successful completion of these studies will establish a method for enhancing the immunogenicity of a previously tested pandemic vaccine. This new vaccine should significantly improve stockpiling capacity and provide an important degree of protection against any newly emerging strains of H5N1. PUBLIC HEALTH RELEVANCE: There is great international concern that the highly pathogenic H5N1 avian influenza virus will cause a pandemic infection resulting in millions of deaths. New vaccine technologies are need if we are to successfully prevent H5N1 infection and avert a global catastrophe. Adjuvants that stimulate dendritic cells can be used to increase vaccine potency, thereby stretching vital manufacturing capacity and protecting against newly emerging strains of H5N1 virus.